Plasma levels of high-density lipoproteins (HDL) are

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Monocyte Inflammatory Response

Andrew J. Murphy, Kevin J. Woollard, Anh Hoang, Nigora Mukhamedova, Roslynn A. Stirzaker, Sally P.A. McCormick, Alan T. Remaley, Dmitri Sviridov, Jaye Chin-Dusting

Objective—Whereas the anti–inflammatory effects of high-density lipoprotein (HDL) on endothelial cells are well described, such effects on monocytes are less studied.

Methods and Results—Human monocytes were isolated from whole blood followed by assessment of CD11b activation/expression and cell adhesion under shear-flow. HDL caused a dose-dependent reduction in the activation of CD11b induced by PMA or receptor-dependent agonists. The constituent of HDL responsible for the antiinflammatory effects on CD11b activation was found to be apolipoprotein A-I (apoA-I). Cyclodextrin, but not cyclodextrin/cholesterol complex, also inhibited PMA-induced CD11b activation implicating cholesterol efflux as the main mechanism. This was further confirmed with the demonstration that cholesterol content of lipid rafts diminished after treatment with the cholesterol acceptors. Blocking ABCA1 with an anti-ABCA1 antibody abolished the effect of apoA-I. Furthermore, monocytes derived from a Tangier disease patient definitively confirmed the requirement of ABCA1 in apoA-I–

mediated CD11b inhibition. The antiinflammatory effects of apoA-I were also observed in functional models including cell adhesion to an endothelial cell monolayer, monocytic spreading under shear flow, and transmigration.

Conclusions—HDL and apoA-I exhibit an antiinflammatory effect on human monocytes by inhibiting activation of CD11b. ApoA-I acts through ABCA1, whereas HDL may act through several receptors. (Arterioscler Thromb Vasc Biol. 2008;28:2071-2077)

Key Words: apolipoprotein A-I 䡲 CD11b 䡲 monocyte 䡲 ABCA1 䡲 Tangier

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lasma levels of high-density lipoproteins (HDL) are inversely associated with cardiovascular morbidity and mortality.^1,2 The most comprehensively studied function of HDL is reverse cholesterol transport. Other cardioprotective functions include its antioxidative properties and its ability to increase nitric oxide (NO) bioavailability.^3,4 More recently, the antiinflammatory effects of HDL, particularly in the endothelium, have been reported.^5,6

See accompanying article on page 1890 A critical event in the formation of atherosclerotic plaques is the recruitment of monocytes into the adventitia where they differentiate into macrophages and ingest modified low- density lipoproteins (LDL) through scavenger receptors to form foam cells.⁷The recruitment of monocytes involves the expression of both endothelial and monocytic adhesion molecules. In the multi-step adhesion cascade the initial monocyte-endothelium attachment occurs via selectins expressed on endothelial cells. Firm adhesion then occurs through vascular cell adhesion molecule (VCAM)-1 and

intracellular adhesion molecule-1 (ICAM-1) interacting with monocyte adhesion molecules such as CD11b/CD18 (Mac-1, CR3).^{8 –10}

A reduction in tumor necrosis factor (TNF)-␣–induced expression of VCAM-1, ICAM-1, and E-selectin in endothelial cells preincubated with HDL has been reported.^11,12 Similarly, decreases in reactive oxygen species production, neutrophil infiltration, and monocyte chemoattractant protein-1 (MCP-1) have also been reported.^13,14It has been demonstrated that inhibition by HDL of E-selectin expression on human endothelial cells is mediated by lysosphingolipids,¹¹ suggesting the involvement of both the scavenger receptor class B-1 (SR-B1) and the S1P3receptor, activating endothelial nitric oxide synthase (eNOS) to produce NO.^3,15,16 The in vivo effects, however, are complex and can be mediated by reconstituted HDL (rHDL) or lipid free apoA- I.^13,14 In contrast, at least to the in vitro findings using endothelial cells, studies on the antiinflammatory effects of HDL on neutrophil activation demonstrate, with one notable exception,¹⁷ that apoA-I is responsible.^{18 –21} Interestingly,

Original received January 2, 2008; final version accepted July 1, 2008.

From the Laboratories of Vascular Pharmacology (A.J.M., K.J.W., J.C.D.) and Lipoproteins and Atherosclerosis (A.H., N.M., D.S.), Baker Heart Research Institute, Melbourne, Victoria, Australia; Molecular Medicine (R.A.S.), The Walter and Eliza Hall Institute of Medical Research, Melbourne, Victoria, Australia; the Department of Biochemistry (S.P.A.M.), University of Otago, Dunedin, Otago, New Zealand; and the Lipoprotein Metabolism Section (A.T.R.), National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Md.

Correspondence to Jaye Chin-Dusting, Baker Heart Research Institute, PO Box 6492, St Kilda Rd Central, Victoria 8008 Australia. E-mail jaye.chin- dusting@baker.edu.au

Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.org DOI: 10.1161/ATVBAHA.108.168690 2071

Downloaded from http://ahajournals.org by on January 20, 2022

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HDL also inhibits oxidized LDL (oxLDL)–induced leukocyte–

endothelial interactions without the induction of endothelial adhesion molecule expression, nor was leukocyte adhesion attenuated by blocking the endothelial adhesion molecules.²²

In this article, we explore the mechanism by which HDL and apoA-I prevents and reverses leukocyte activation. Our study shows that HDL and apoA-I act through various receptors to decrease monocyte activation and that the major contributing pathway for apoA-I is the monocytic ATP- binding cassette transporter A1 (ABCA1).

Methods

Full details are provided in the supplemental materials (available online at http://atvb.ahajournals.org.

Study Subjects: Healthy and R1068H Tangier Family

The study was approved by the Human Ethics committees of the Alfred Hospital and the University of Otago; informed consent was obtained from all donors. Blood was anticoagulated with sodium citrate (19.2mMol/L) or EDTA-vacutainer tubes (Tangier study).

Monocyte Isolation

Resting human monocytes were isolated by density centrifugation with Lymphoprep followed by Dynal Negative Monocyte Isolation kit as described previously.²³

Cholesterol Acceptors

HDL was isolated from plasma using sequential ultracentrifugation (density 1.085 to 1.21g/mL), and protein content was measured.

Reconstituted HDL (rHDL)^24,25and phosphatidylcholine liposomes were prepared as previously described²⁶; all HDL treatments were performed using 50␮g/mL unless otherwise stated. Human plasma apoA-I was isolated as previously described²⁷and used at 40␮g/mL.

Beta-cyclodextrin and cholesterol saturated cyclodextrin was prepared as previously described.²⁸The L37pA peptide was synthesized as described.²⁹

Receptor Blocking and Trapping Studies

Monocyte receptors were blocked using specific blocking antibodies for 4 hours at 4°C. Antimouse IgM (Sigma) was used as a control Ab.

Flow Cytometry

Monocytes were stimulated and incubated with the fluorescein isothiocyanate (FITC)-Ab to CD11b for 15 minutes at 37°C, unless otherwise stated. Cells were fixed and CD11b expression was measured by flow cytometry. Samples were controlled for by using the isotype matched negative control. Results were expressed as percentage of the unstimulated control (100%) or PMA (100%, Tangiers only). For lipid raft quantification monocytes were treated for 15 minutes at 37°C, centrifuged, and incubated with FITC- Cholera toxin B (CT-B) for 1 hour at room temperature and rafts measured by flow cytometry.

Lipid Raft Staining

Rafts were stained using the Vybrant lipid raft labeling kit as per the manufacturer’s instructions. Monocytes were mounted in fluorescence mounting media and viewed on the fluorescent microscope.

Staining intensity was quantified using Image Pro software.

Perfusion Studies

Perfusion Studies were conducted using the parallel plate flow- chamber as previously described.³⁰Prestimulated monocytes were perfused over human coronary aortic endothelial cells (HCAECs) at a shear-rate of 150s^⫺1(1.1dyn/cm²) for 5 minutes with a washout period of 5 minutes. Monocyte adhesion was captured and analyzed offline.

Monocyte Spreading/Adhesion Perfusion Assay

Perfusion studies were conducted in platelet coated glass microcap- illary tubes at 37°C.³¹Preactivated monocytes were perfused over the platelet monolayer for 5 minutes (t⫽0 seconds) followed by a washout period of 5 minutes (t⫽300 seconds). Monocyte-platelet interactions were visualized according to “perfusion studies.”

Static Adhesion Assay

Monocyte adhesion to immobilized fibrinogen was performed for 15 minutes at 37°C as previously described.³¹

Migration Assay

Migration assays were performed using 8.0 ␮mol/L Transwells.³² Monocytes with treatment were seeded in the upper chamber, and allowed to migrate for 30 minutes at 37°C to the lower chamber containing 50 ng/mL of MCP-1. Migrated monocytes were fixed and the number of migrated cells quantified.

Filamentous Actin Content

Monocytes were stained for F-actin with alexa fluor 488-Phalloidin and quantified by flow cytometry or further stained with DAPI and investigated by confocal microscopy.

Statistical Analysis

Values are presented as the mean⫾SEM or percentage of control⫾SEM. All results were analyzed for statistical significance using 1-way ANOVA followed by Bonferroni posthoc test, except Perfusion studies which were analyzed using a 2-way ANOVA followed by Bonferroni posthoc test. Significance was accepted at P⬍0.05.

Results

HDL Inhibits PMA-Induced Activation of CD11b PMA induced monocytic integrin CD11b activation which was dose-dependently inhibited by HDL (2 to 50 ␮g/mL;

Figure 1A). Although the HDL concentrations used in this study are below plasma levels, they are approaching saturat- ing concentrations described in cholesterol efflux experi- ments, routinely used by others.³³The decrease in activated CD11b was accompanied by a decrease in total CD11b abundance (PMA versus HDL (50␮g/mL) ⫹ PMA: 19⫾1.84 versus 9.7⫾1.89 U; n⫽4, P⬍0.001).

To assess whether the response to HDL was dependent on monocyte heterogeneity, CD16⫹ and CD16- monocytes were isolated and their response compared. There was no difference between the two subsets in response to HDL (supplemental Figure I).

ApoA-I Reduces CD11b Activation

Reconstituted HDL and apoA-I inhibited CD11b activation to a similar extent to HDL (Figure 1B). In contrast, neither BSA nor phosphatidylcholine liposomes had any effect (Figure 1A and 1B). HDL and apoA-I significantly reduced both lipopolysaccharide (LPS) and fMLP-induced CD11b activation (supplemental Figure II).

HDL Prevents and Reverses Monocyte Activation Pretreatment of monocytes with HDL followed by stimulation with either PMA (Figure 1C) or LPS (LPS versus Prevention; 152⫾1.8 versus 101⫾4.5, n⫽5, P⬍0.01) led to a significant reduction of CD11b expression (Figure 1C).

Likewise, prestimulation of monocytes with PMA followed by a 15-minute incubation with HDL also significantly

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reduced CD11b expression (Figure 1C), also seen with LPS (LPS versus Reversal; 152⫾1.8 versus 92.8⫾5.7, n⫽5, P⬍0.01). Monocytes preincubated with HDL or apoA-I, washed, and then challenged with either PMA (or fMLP, data not shown) also demonstrated prevention of monocytes from activation (Figure 1D).

Regulation of CD11b Expression and Cholesterol Efflux

Cyclodextrin significantly attenuated PMA-induced CD11b activation (Figure 2A). Cholesterol-saturated cyclodextrin (Ch-CD) had no effect on PMA-induced CD11b activation (Figure 2A).

Changes in Monocyte Lipid Raft Abundance Treatment of monocytes with apoA-I, HDL, and CD, but not liposomes and BSA, significantly decreased lipid rafts in the plasma membrane (Figure 2B). Incubation with apoA-I dra- matically modified membrane raft abundance (control versus apoA-I; 66.9⫾11.3 relative fluorescence units [rfu] versus 37.3⫾4.6 rfu, n⫽5, P⬍0.05; represented visually in Figure 2C), indicating rapid efflux from plasma membrane rafts.

Involvement of SR-B1

Blocking SR-B1 blunted the effect of HDL on CD11b activation, albeit not significantly (P⫽0.18; Figure 3A).

SR-B1 blockade failed to affect the inhibition of CD11b activation induced by apoA-I (Figure 3A). An irrelevant isotype matched Ab (cAb) had no effect.

Involvement of ABCA1

The antiinflammatory effects of apoA-I but not HDL (Figure 3B) were abolished in the presence of the ABCA1 blocking antibody NDF4C2. Irrelevant isotype matched Ab showed no effect. The role for ABCA1 internalization was examined using NDF6F1,³⁴which does not affect ABCA1-dependent cholesterol efflux but prevents ABCA1 internalization and degradation. NDF6F1 had no effect on the inhibitory action of apoA-I on PMA-induced CD11b expression (Figure 3C).

Monocyte Adhesion to Endothelial Cells Under Shear-Flow

Compared to unstimulated monocytes, PMA significantly increased monocyte adhesion to endothelial cells. Coincuba- tion of monocytes with PMA and HDL or apoA-I resulted in a significant reduction in monocyte adhesion (Figure 4A and 4D). The importance of the interaction between apoA-I and Figure 1. CD11b activation. Monocytes were stimulated with (A)

PMA⫾HDL or BSA (B) PMA⫾rHDL, apoA-I, or liposomes. In prevention and reversal studies (C) monocytes were preincubated with HDL followed by PMA or vice versa. (D) Monocytes were stimulated after preincubation with HDL or apoA-I which were removed.

Figure 2. Cholesterol efflux. (A) Monocytes were incubated with PMA⫾cyclodextrin (CD) or cholesterol saturated cyclodextrin (CD-CH).

(B) Monocytes were incubated with apoA-I, HDL, CD, and BSA for 15 minutes, lipid rafts stained with CT-B, and cells analyzed by flow cytometry. (C) Confocal microscopy image of monocytes stained with CT-D without (top panel) and with treatment with apoA-I.

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ABCA1 was also examined under shear flow. A significant apoA-I–induced reduction in adhesion was no longer evident in the presence of the ABCA1 antibody (Figure 4E). NDF4C2 alone or an isotype matched Ab had no effect on adhesion (data not shown).

Monocyte Spreading and Adhesion on Platelets Under Shear-Flow

To explore the effect of HDL on monocyte spreading under shear flow conditions, PMA stimulated monocytes were perfused over a platelet monolayer for 5 minutes (time⫽0 seconds) followed by a 5-minute washout period (time⫽300

seconds; with or without HDL). Before washout there was no difference in cell spreading between the 2 groups. Washing of stimulated monocytes with HDL-containing buffer resulted in a significant reduction in spreading compared to washout with buffer alone (Figure 5A). A significant reduction in monocyte adhesion to platelets was also observed after washout with HDL compared to buffer alone (supplemental Figure III).

Monocyte Transmigration

MCP-1 facilitated a significant monocytic migratory response, which was markedly reduced when the monocytes were preincubated with either HDL or apoA-I (Figure 5B).

Monocyte F-Actin Content

Stimulation of monocytes with PMA resulted in increased F-actin levels, which was significantly reduced by coincuba- tion with HDL and PMA (Figure 5C). This observation was confirmed by flow cytometry (P⬍0.01; Figure 5D).

Figure 3. Blocking studies. (A) Monocytes incubated with SR-B1 blocking or control antibody (cAb) were treated with PMA⫾apoA-I or HDL. (B) Monocytes incubated with NDF4C2 anti-ABCA1 Ab(4C2) or cAb were treated with PMA⫾apoA-I or HDL or (C) the NDF6F1 anti-ABCA1 Ab (6F1).

Figure 4. Monocyte adhesion under flow. (A) Monocytes were pretreated with PMA (F), PMA⫹HDL (E), or PMA⫹apoA-I () before perfusion over HCAECs. Images of cell adhesion after 5 minutes; with PMA (B) PMA⫹HDL (C) and PMA⫹apoA-I (D). (E) Monocytes were treated with PMA (F), PMA⫹apoA-I (E), and NDF4C2 anti-ABCA1 Ab with PMA⫹apoA-I ().

Figure 5. Monocyte phenotype. Monocyte spreading (A) under shear-flow⫾HDL (E). Monocyte transmigration. (B) Monocytes (⫾treatment) were seeded in the upper chamber of a transwell and allowed to migrate toward MCP-1. F-actin content. (C) Con- focal images of resting monocytes⫾PMA ⫾HDL stained for F-actin (green) and nuclei (blue). (D) F-actin content measured using phalloidin.

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L37pA Inhibits Monocyte Activation Similar to ApoA-I

The apoA-I mimetic peptide L37pA²⁹ (10 and 20 ␮g/mL) significantly reduced PMA stimulated CD11b expression on monocytes as well as PMA challenged monocyte adhesion to fibrinogen coated glass cover-slips under static conditions (supplemental Figure IV).

Tangier Patient Derived Monocytes

For simplification of comparison between members of the R1068H Tangier family, results are expressed as percentage of CD11b expression after activation with PMA. Monocytes isolated from an unaffected relative, treated with PMA and apoA-I or HDL, displayed a similar reduction to that observed in previous assays (Figure 6A). Heterozygote monocytes had a diminished ability to respond to apoA-I, however a reduction in CD11b activation was still observed. Mono- cytes derived from the Tangier disease patient failed to respond to apoA-I treatment. Although there was a clear gene-dose-dependent response to apoA-I, HDL reduced CD11b activation similarly in both heterozygote and Tangier derived monocytes (Figure 6A). Similar results were obtained when assessing the ability of both apoA-I and HDL to reduce the adhesion of monocytes to fibrinogen (Figure 6B).

Discussion

The activation of monocytes is a pivotal event in vascular inflammation and atherosclerosis. In the current study we report that HDL and apoA-I can prevent as well as reverse the activation of human monocytes with apoA-I exerting its effects through ABCA1.

The main finding of this article is that HDL dose- dependently decreases CD11b expression and activation on primary human monocytes stimulated with PMA. This finding was also observed with the receptor-mediated activators LPS and fMLP.^23,35,36 Both rHDL and lipid-free apoA-I inhibited PMA-induced activation of CD11b, however phos- pholipid liposomes or albumin had no effect. This is consistent with previous findings where apoA-I inhibited monocyte spreading over time in response to M-colony stimulating factor (CSF).²¹Interestingly, the mechanism by which HDL and apoA-I inhibit monocyte activation appears to be different from that of the antiinflammatory actions of these

molecules on endothelial cells, thought to be mediated via HDL-stimulating NO production.^11,16,37

Cholesterol efflux appears to be a requirement for the effects of apoA-I and HDL because cyclodextrin effectively mimicked HDL and apoA-I in inhibiting CD11b activation on monocytes. Although cyclodextrin removes cholesterol non- specifically,³⁸it removes it from the same plasma membrane pools as apoA-I as evidenced by the enhanced efflux to cyclodextrin after overexpression of ABCA1.³⁹Loading of cyclodextrin with cholesterol, which converts cyclodextrin to a cholesterol donor, rendered it inactive thus confirming that cholesterol efflux is required. Interestingly cyclodextrin and cholesterol removal has been shown to inhibit monocyte spreading when coincubated with M-CSF²¹and cause rapid retraction of membrane protrusions of macrophages.⁴⁰Fur- ther we demonstrated that short incubations with apoA-I, HDL, and CD, but not liposomes and BSA, resulted in depletion of raft cholesterol. It may be that the latter do not incite perturbation as potently as the former cholesterol acceptors. Regardless, the above findings suggest that rapid depletion of lipid from cell membranes appears to be a key mechanism influencing the inflammatory response of the monocyte/macrophage.

To examine the specific mechanisms connecting the effects of HDL on monocyte activation and cholesterol efflux, we investigated the involvement of 2 HDL receptors, SR-B1 and ABCA1. SR-B1, along with ABCG1, has been shown to be involved in supporting cholesterol efflux to HDL, whereas lipid-poor apoA-I removes cholesterol exclusively through ABCA1-dependent pathways.⁴¹Blocking SR-B1 resulted in a reduction, but not elimination, of the antiinflammatory effects of HDL, however it did not affect apoA-I. Blocking ABCA1 totally abolished the inhibitory effect of apoA-I but had no effect on HDL. Thus, the apoA-1/ABCA1 interaction is likely to be a major pathway mediating these effects, with other pathways, such as SR-B1 and ABCG1, also contributing to the effects of HDL.

Shear flow adhesion assays were used to examine the functional outcome of HDL in reducing monocyte activation.

When either HDL or apoA-I was present, PMA-stimulated monocyte adhesion to HCAECs was significantly attenuated.

Furthermore, blocking monocyte ABCA1 reverses the apoA- I–induced decrease in adhesion, consistent with the results of the CD11b activation assay and confirming the involvement of ABCA1.

Although it has been previously demonstrated that HDL can inhibit monocyte spreading,²¹ this is the first report describing this effect under physiological shear conditions.

The inhibition of the cell adhesion cascade by HDL was further investigated by examining the effects of HDL and apoA-I on monocyte migration to MCP-1. Both HDL and apoA-I were able to significantly inhibit monocyte migration to MCP-1, a finding consistent with the observations of Navab et al.⁴² Because changes in the cytoskeleton are required to induce spreading and migration of cells which has been shown to be associated with an increase in F-actin content,⁴³ total F-actin was quantified and demonstrated to decrease in the presence of HDL. This finding is consistent with the decrease in spreading observed by Diederich et al.²¹ Figure 6. Tangier monocytes. (A) Monocytes from individuals

(white⫽unaffected, gray⫽hetero, black⫽Tangier) were stimulated with PMA⫾apoA-I or HDL and CD11b activation measured. (B) Monocytes treated with PMA⫾apoA-I or HDL were added to fibrinogen where adherence was quantified.

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The inhibitory effect of HDL on monocytes was effective in both preventative and reversal settings. The latter finding, in particular, has important implications in disease states such as acute coronary syndrome, where the process of inflammation has already occurred. Here we have clearly demonstrated that prestimulated monocytes can be rescued from activation by HDL. This may further explain the findings of previous studies demonstrating that HDL/apoA-I can reduce the activation of the endothelium and neutrophils accumulated in the intima-media in an in vivo inflammatory model up to 9 hours postinjury.¹⁴ Our findings are also consistent with animal studies where the short term elevation of HDL administration was atheroprotective.⁴⁴

Recent termination of CETP inhibition trials suggest different strategies for raising HDL levels are required.⁴⁵ The development of apoA-I mimetics may provide an effective alternative. L37pA has previously been shown to effectively stimulate cholesterol efflux and stabilization of ABCA1 through the same mechanisms as apoA-I.^29,46,47In our studies L37pA was able to mimic apoA-I antiinflammatory actions on monocytes indicating a potential for peptide based thera- peutics in inflammatory diseases.

Finally, we explored the antiinflammatory role of HDL and apoA-I in a Tangier disease subject along with a heterozygote subject and an unaffected member from the R1068H family.⁴⁸ Tangier disease patients have a dysfunctional ABCA1 unable to support cholesterol efflux to apoA-I.⁴⁹ The response of monocytes from the unaffected family member was similar to that of healthy subjects, monocytes from the heterozygote subject responded to both HDL and apoA-I albeit less than compared to monocytes of unaffected family member. In contrast, apoA-I did not decrease CD11b activation in monocytes of the Tangier disease patient, although HDL still produced a degree of inhibition. Similar effects were observed when examining adhesion of monocytes of Tangier disease patient to fibrinogen. These findings are consistent with our hypothesis that apoA-I is working via ABCA1 to inhibit monocytic activation, whereas HDL additionally en- gages via an ABCA1-independent pathway.

In summary, this study details for the first time the mechanism by which HDL and apoA-I regulate monocyte adhesion, spreading, and integrin activation. The finding that apoA-I is equally potent to HDL in inhibiting elements of inflammation provides important insight into the development of novel strategies such as apoA-I mimetic peptides in the treatment and control of atherosclerosis.⁵⁰ The finding that HDL can still influence the inflammatory status of monocytes from Tangier disease patients also indicates that therapeutic HDL strategies can be applied in the patient population which is at risk of CVD. The ability of HDL to prevent and reverse activation of monocytes may also be of significant interest for management a variety of inflammatory diseases.

Acknowledgments

We acknowledge Rachel Brace for her efforts in helping with the blood collection and providing medical care for the R1068H family.

Sources of Funding

A.J.M. is supported by an industry scholarship form Actelion Ltd, Sydney. K.J.W. is an Australian National Heart Foundation Research Fellow. D.S. and J.C.-D. are Senior Research Fellows of the Australian National Health and Medical Research Council.

Disclosures

None.

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